Vesicular stomatitis virus (VSV) is a single-stranded RNA virus belonging to the same family (Rhabdoviridae) as rabies virus. Virions contain an RNA polymerase (transcriptase) which makes messenger RNAs which are complementary to genomic RNA. The mechanism whereby monocistronic, capped, methylated, polyadenylated mRNAs are generated is not understood. Three viral proteins (N, L, and NS) are required and host proteins may be involved. It is likely that replication involves the same viral proteins. We plan to use temperature-sensitive mutants of VSV to elucidate the mechanism of transcription and whether or not modifications (e.g., capping) are virally coded. We will examine temperature-sensitive mutants of VSV, Indiana serotype, especially those belonging to group IV, but also those belonging to group II, to determine the identify of the defective protein. This will be done by methods involving protein purification and reconstitution of transcriptase activity using homologous and heterologous systems, as well as by using techniques such as two-dimensional gel electrophoresis of proteins and examination of the digestion products of partial or complete protease digestions to locate the mutant protein. We plan to investigate the RNA produced in vitro by a variety of mutants of complementation groups I (probably representing L protein mutants), II and IV in the hope that aberrant patterns of RNA synthesis in such mutants may reveal more of the nature of the transcription process and the role of viral proteins in that process. Since transcription and replication are closely related processes, such experiments may enable us to propose hypotheses for the mechanism of replication.